Artikel Abdullah UNDIP 8
Reaktor, Vol. 11 No.1, Juni 2007, Hal. : 50-52
SOLID AND LIQUID PINEAPPLE WASTE UTILIZATION
FOR LACTIC ACID FERMENTATION
USING Lactobacillus delbrueckii
Abdullah*)
Abstract
The liquid and solid pineapple wastes contain mainly sucrose, glucose, fructose and other nutrients. It
therefore can potentially be used as carbon source for fermentation to produce organic acid. Recently,
lactic acid has been considered to be an important raw material for production of biodegradable
lactate polymer. The experiments were carried out in batch fermentation using the liquid and solid
pineapple wastes to produce lactic acid. The anaerobic fermentation of lactic acid were performed at
40 oC, pH 6, 5% inocolum and 50 rpm. Initially results show that the liquid pineapple waste by using
Lactobacillus delbrueckii can be used as carbon source for lactic acid fermentation. The production
of lactic acid are found to be 79 % yield, while only 56% yield was produced by using solid waste.
Keywords : Lactic acid fermentation, Pineapple waste, Lactobacillus delbrueckii.
Introduction
Pineapple canning industries located in tropical
region such as Indonesia, Malaysia and Thailand
produce large quantity of solid and liquid waste. In
canneries, nearly 75% of the fruit in the form of peeled
skin, core, crown end, etc. is no utilized and is
discharged as wastage causing problems of disposal and
pollution. The dry matter content of pineapple waste is
around 10%, composed of about 96% organic matter
and 4 % inorganic matter (Chandapillai and Selvarajah,
1978). The waste contain high concentration of
biodegradable organic material and suspended solid. As
a result it has a high BOD and extremes of pH
conditions (Buckle, 1989). Beside their pollution and
hazard aspects in many cases, food processing waste
such as pineapple waste might have a potential for
recycling to get raw material or for conversion into
useful product of higher value added products, or even
as raw material for other industries, or for use as food or
feed after biological treatment (Kroyer, 1991).
Lactic acid is produced from mono or disacharides via the Embden-Meyerhof glycolysis. Under
anaerobic conditions the pyruvic acid so produced and
than is reduced to lactic acid by the enzyme lactic
dehydrogenises. Glucose, maltose, lactose, and sucrose
from cheap raw materials such as molasses, whey, cane
and beat sugar and starch wastes have been used as
substrate for fermentation process to produce lactic acid
(Milson, 1987). The lactic acid production in the world
about 40.000 ton per year, which more than 50% is
utilized in food technology as pH regulator, microbial
preservative or buffering agent. In the chemical
industry, lactic acid is used in as solvent or pH
regulation, and as raw material for biodegradable
polymer (Parajo et al., 1996).
*)
In industrial of lactic acid production by
fermentation, the carbohydrate containing medium
has been enriched with nutrient such as yeast extract
and malt sprout to achieve optimal growth conditions
for the demand of nutrient lactic acid bacteria
(Atkinson and Mavituna, 1991). Based on the
characteristic of pineapple waste, the liquid waste has
small nitrogen and soluble protein contents (Abdullah
and Mat, 1998). Therefore the substrate must be
supplemented by yeast extract as a nitrogen source
In the present study, liquid and solid pineapple
waste was used as substrate. Lactobacillus
delbrueckii was used to produce lactic acid by
fermentation. The fermentation was carried out in
batch mode, with working volume 1 liter and pH,
temperature and speed were controlled at 6.0, 40oC
and 50 rpm.
Materials And Methods
Substrate
The fermentation media containing liquid and
solid pineapple waste was obtained from Malaysian
Cannery of Malaysia Sdn. Bhd. The pre-treatment of
substrate was described by Lazaro (1989).
Strain
The micro-organism used in this study was
Lactobacillus delbrueckii subsp. delbrueckii ATCC
9649 obtained from DSMZ, Germany. The strain was
maintained on MRS agar at 4oC and transferred to
fresh medium every month.
Inoculum Media
Each fermentation process was initiated by
transferring a small amount of biomass to a 250ml
Erlenmeyer flask containing 50ml of liquid MRS
Chemical Engineering Department, Diponegoro University
Jl. Prof. Sudarto, SH, Tembalang-Semarang 50239; Telp.(024) 7460058
50
Solid and Liquid Pineapple Waste …
Dry Cell Weight
Cell concentration was measured by constructing
a calibration curve of optical Density as a function of
dry cell weight. Dry weight determined by
centrifugation at 4000 rpm for 15 minute, washing
twice with distilled water and dried at 103oC for 24
hours and weighing (Aeschlimann, and Stockar, 1987).
The optical density was measured on spectrophotometer
(Shimadzu UV-1601) at 620 nm.
Chemical Analysis
The Organic acid content was measured by
HPLC (Waters TM 600). A 250 mm X 4.6 mm ID
Spherisob Octyl column (Waters) with UV detector
(210 nm) was used. The eluent used was 0.2 M
phosphoric acid at flow rate 0.8 ml per minute and
temperature 25 oC. The sugar content was also
measured by the same HPLC, using a 300 mm X 4 mm
ID.µ Bondapak/Carbohydrate column (Waters) with RI
detector. The eluent used was a mixture acetonitrile :
water (80:20) at flow rate 2 ml per minute and
temperature 25 oC.
Results And Discussion
Lactic acid production using liquid waste
The pineapple waste is a complex medium
containing saccharides, the primary species of fructose,
glucose and sucrose accounting for most of the
carbohydrate content available for fermentation. The
individual sugar utilization during fermentation of
pineapple waste using L. delbrueckii can be seen in Fig.
1.
Sucrose was consumed completely in 62 h.
Before fermentation process a few organisms having
phosphorolytic enzymes phosphorylyze the disacharide
51
60
2.0
50
1.5
40
Glucose
Fructose
30
Succrose
1.0
L.Acid
Biomass
20
0.5
Biomass concentration (g/l)
Batch Fermentation
The fermentation was carried out in 3-liter
fermentor (Biostat B Model). The fermentor was
equipped with pH, temperature and dissolved oxygen
controllers. The fermentor containing 950 ml substrate
was first sterilized at 121oC for 15 minutes. 50 ml of
Inocolum was sterilized separately and added
aseptically to the fermentor. Anaerobic system were
produced by sparged the fermentor by nitrogen 6.5
ml/minute and speed at 50 rpm (Lund et al., 1992).
Samples of 10-20ml were withdrawn from the
fermentation broth at regular time interval. The
microbial cells were separated by centrifugation for dry
biomass determination. The supernatant was
immediately frozen for further determination of the
lactic acid, glucose, fructose and sucrose concentrations
(Mercier and Yerushalmi , 1992).
to glucose-1-phosphate and fructose. The sucrose was
also by acidic condition, to glucose and fructose
(Zubay, 1984), so the concentration of glucose and
fructose increase. This indicates that the rate of
hydrolysis is faster than fermentation process. The
glucose consumption is better than fructose present in
medium, but both sugars were not completely
utilized. This may reflect the complex nature of
pineapple waste which inhibit the fermentation
process.
The production of lactic acid after 7 days is
54.3 g/l or 79% yield based of the total sugar.
Lactic acid and sugar concentration (g/l)
medium. Anaerobic condition was produced by flushing
with nitrogen and sealing them with thigh-fitting rubber
stopper (Sakamoto and Komagata, 1996). The inoculum
was shaken in incubator shaker at 37 oC, 150 rpm for 24
hours.
(Abdullah)
10
0
0
100
200
0.0
300
Tim e (h)
Figure 1. The Time dependence of biomass, sugar
and lactic acid concentration during fermentation of
liquid waste. Experimental conditions: T: 40oC; pH:
6; 5% inoculum and 50 rpm.
Lactic acid production using solid waste
Solid waste (10%) is extracted by water for
one hour in Erlenmeyer shaker at 250 rpm and room
temperature. The solid particles were separated by
vacuum filter and the liquid was used to fermentation
process in fermentor at 40oC, pH 6, 50 rpm and
inoculum 5%. The initial sugar concentrations about
23.33 g/l of sugar consist only glucose and fructose.
After 6 days the concentration of sugar decreases
from 10.26 to 1.24 g/l and fructose from 12.84 to
4.45 g/l (Fig.2). This indicates that glucose utilization
better than fructose present in medium, but both is
sugars were not completely utilized (87.9 % and
65.34%). The lactic acid production is only 13.1 g/l
or 56% yield. This may reflect that the complex
nature of waste inhibits the fermentation process.
Reaktor, Vol. 11 No.1, Juni 2007, Hal. : 50-52
Rogers P.L. Breach Science Publishers. New York.
pp: 261-277.
16
14
Chandapillai, M.M. and .Selvarajah T., (1978), ”The
Potential Use of Agricultural Waste in Livestock
Production in Malaysia”, Food and Agriculture
Malaysia 2000, Ed. Chin H.F., Faculty of Agriculture
UPM Malaysia. pp: 257-274. 1978.
Concentration (g/l)
12
10
Biomass
Glucose
8
Kroyer, G.T., (1991), “Food Processing Wastes”,
Bioconversion of Waste Material to Industrial
Product. Ed. Martin, A.M. Elviser Applied Science
London. pp:293-303.
Fructose
L. Acid
6
4
Lazaro, M.J., (1989), “Liquid Chromatographic
Determination of Acid and Sugar in Homolactic
Cucumbar Fermentation”, Journal AOAC. 72(1): pp
52-55.
2
0
0
100
200
300
Time (h)
Figure 2. The time dependence of sugar and lactic acid
concentration during fermentation of liquid extract.
Experimental conditions: T: 40oC, pH: 6,5% inoculum
and 50 rpm. .
Conclusions
The chemical composition of the pineapple waste
appears to be a good nutrient for cultivation of lactic
acid bacteria. It can potentially be used as carbon source
for lactic acid fermentation. The yield of lactic acid
from liquid are found to be 79 % yield, while only 56%
yield was produced from solid waste.
References
Abdullah, M. Busairi, and Mat, H.B., (1998), “Pencirian
Bahan Sisa Nanas Tempatan”, Simposium Kimia
Analisis ke Sebelas, Universiti Technologi Malaysia.
Aeschlimann, A. and Stockar, U.V., (1987), “The
Production of Lactic Acid from Whey Permeate by L.
helviticus.” Abstract of Fourth European Congress on
Biotechnology, 2: pp 132-135.
Atkinson ,B., and F. .Mavituna, (1991), ”Biochemical
Engineering and Biotechnology Hand Book“ 2nd.
Edition , Stockton Press, New York, 1181 - 1183.
Buckle, K.A., (1989), “Biotechnology Opportunities in
Waste Treatment and Utilization for The Food
Industry”, Biotechnology and The Food Industry, Ed.
Lund, B., Norddahl, B and Ahring, B., (1992),
“Production of Lactic Acid from Whey Using
Hydrolysed Whey Protein as Nitrogen Source”,
Biotechnology Letters. 14(9): pp : 851-856
Mercier, P. and Yerushalmi, L., (1992), “Kinetics of
Lactic Acid Fermentation on Glucose and Corn by L.
amylophilus”, J. Chem Tech. Biotechnology . 55 : pp
111 - 121.
Milson, P.E., (1987), “Organic Acid by Fermentat
ion”, The Food Biotechnology, Ed. King, R.D.
Elsevier Applied Science. London. pp: 294-298.
Parajo, C.Alonso, J.L and Santos,V., (1996), “Lactic
Acid from Wood”, Process Biochemestry. 31(3).
pp:271-280.
Sakamoto,M. and Komagata, K., (1996), “Aerobic
Growth of An Activities of NADH Oxydase and
NADH Peroxidase in Lactic Acid Bacteria”, J.
Fermentation and Bioengineering, 29 : pp 591 - 602.
Sasaki, K., Noparatnaraphorn, N. and Nagai. S.,
(1991), “Use of Photosynthetic Bacteria for The
Production of SCP and Chemicals from Agro
Industrial Waste”, Bioconversion of Waste Material
to Industrial Product, Ed. Martin. A.M. Elviser
Applied Science. London. pp 225-233.
Zubay, G.L., (1984), “Biochemestry”, USA:
Addison-Wesley Publiushing Company. pp 283-290.
52
SOLID AND LIQUID PINEAPPLE WASTE UTILIZATION
FOR LACTIC ACID FERMENTATION
USING Lactobacillus delbrueckii
Abdullah*)
Abstract
The liquid and solid pineapple wastes contain mainly sucrose, glucose, fructose and other nutrients. It
therefore can potentially be used as carbon source for fermentation to produce organic acid. Recently,
lactic acid has been considered to be an important raw material for production of biodegradable
lactate polymer. The experiments were carried out in batch fermentation using the liquid and solid
pineapple wastes to produce lactic acid. The anaerobic fermentation of lactic acid were performed at
40 oC, pH 6, 5% inocolum and 50 rpm. Initially results show that the liquid pineapple waste by using
Lactobacillus delbrueckii can be used as carbon source for lactic acid fermentation. The production
of lactic acid are found to be 79 % yield, while only 56% yield was produced by using solid waste.
Keywords : Lactic acid fermentation, Pineapple waste, Lactobacillus delbrueckii.
Introduction
Pineapple canning industries located in tropical
region such as Indonesia, Malaysia and Thailand
produce large quantity of solid and liquid waste. In
canneries, nearly 75% of the fruit in the form of peeled
skin, core, crown end, etc. is no utilized and is
discharged as wastage causing problems of disposal and
pollution. The dry matter content of pineapple waste is
around 10%, composed of about 96% organic matter
and 4 % inorganic matter (Chandapillai and Selvarajah,
1978). The waste contain high concentration of
biodegradable organic material and suspended solid. As
a result it has a high BOD and extremes of pH
conditions (Buckle, 1989). Beside their pollution and
hazard aspects in many cases, food processing waste
such as pineapple waste might have a potential for
recycling to get raw material or for conversion into
useful product of higher value added products, or even
as raw material for other industries, or for use as food or
feed after biological treatment (Kroyer, 1991).
Lactic acid is produced from mono or disacharides via the Embden-Meyerhof glycolysis. Under
anaerobic conditions the pyruvic acid so produced and
than is reduced to lactic acid by the enzyme lactic
dehydrogenises. Glucose, maltose, lactose, and sucrose
from cheap raw materials such as molasses, whey, cane
and beat sugar and starch wastes have been used as
substrate for fermentation process to produce lactic acid
(Milson, 1987). The lactic acid production in the world
about 40.000 ton per year, which more than 50% is
utilized in food technology as pH regulator, microbial
preservative or buffering agent. In the chemical
industry, lactic acid is used in as solvent or pH
regulation, and as raw material for biodegradable
polymer (Parajo et al., 1996).
*)
In industrial of lactic acid production by
fermentation, the carbohydrate containing medium
has been enriched with nutrient such as yeast extract
and malt sprout to achieve optimal growth conditions
for the demand of nutrient lactic acid bacteria
(Atkinson and Mavituna, 1991). Based on the
characteristic of pineapple waste, the liquid waste has
small nitrogen and soluble protein contents (Abdullah
and Mat, 1998). Therefore the substrate must be
supplemented by yeast extract as a nitrogen source
In the present study, liquid and solid pineapple
waste was used as substrate. Lactobacillus
delbrueckii was used to produce lactic acid by
fermentation. The fermentation was carried out in
batch mode, with working volume 1 liter and pH,
temperature and speed were controlled at 6.0, 40oC
and 50 rpm.
Materials And Methods
Substrate
The fermentation media containing liquid and
solid pineapple waste was obtained from Malaysian
Cannery of Malaysia Sdn. Bhd. The pre-treatment of
substrate was described by Lazaro (1989).
Strain
The micro-organism used in this study was
Lactobacillus delbrueckii subsp. delbrueckii ATCC
9649 obtained from DSMZ, Germany. The strain was
maintained on MRS agar at 4oC and transferred to
fresh medium every month.
Inoculum Media
Each fermentation process was initiated by
transferring a small amount of biomass to a 250ml
Erlenmeyer flask containing 50ml of liquid MRS
Chemical Engineering Department, Diponegoro University
Jl. Prof. Sudarto, SH, Tembalang-Semarang 50239; Telp.(024) 7460058
50
Solid and Liquid Pineapple Waste …
Dry Cell Weight
Cell concentration was measured by constructing
a calibration curve of optical Density as a function of
dry cell weight. Dry weight determined by
centrifugation at 4000 rpm for 15 minute, washing
twice with distilled water and dried at 103oC for 24
hours and weighing (Aeschlimann, and Stockar, 1987).
The optical density was measured on spectrophotometer
(Shimadzu UV-1601) at 620 nm.
Chemical Analysis
The Organic acid content was measured by
HPLC (Waters TM 600). A 250 mm X 4.6 mm ID
Spherisob Octyl column (Waters) with UV detector
(210 nm) was used. The eluent used was 0.2 M
phosphoric acid at flow rate 0.8 ml per minute and
temperature 25 oC. The sugar content was also
measured by the same HPLC, using a 300 mm X 4 mm
ID.µ Bondapak/Carbohydrate column (Waters) with RI
detector. The eluent used was a mixture acetonitrile :
water (80:20) at flow rate 2 ml per minute and
temperature 25 oC.
Results And Discussion
Lactic acid production using liquid waste
The pineapple waste is a complex medium
containing saccharides, the primary species of fructose,
glucose and sucrose accounting for most of the
carbohydrate content available for fermentation. The
individual sugar utilization during fermentation of
pineapple waste using L. delbrueckii can be seen in Fig.
1.
Sucrose was consumed completely in 62 h.
Before fermentation process a few organisms having
phosphorolytic enzymes phosphorylyze the disacharide
51
60
2.0
50
1.5
40
Glucose
Fructose
30
Succrose
1.0
L.Acid
Biomass
20
0.5
Biomass concentration (g/l)
Batch Fermentation
The fermentation was carried out in 3-liter
fermentor (Biostat B Model). The fermentor was
equipped with pH, temperature and dissolved oxygen
controllers. The fermentor containing 950 ml substrate
was first sterilized at 121oC for 15 minutes. 50 ml of
Inocolum was sterilized separately and added
aseptically to the fermentor. Anaerobic system were
produced by sparged the fermentor by nitrogen 6.5
ml/minute and speed at 50 rpm (Lund et al., 1992).
Samples of 10-20ml were withdrawn from the
fermentation broth at regular time interval. The
microbial cells were separated by centrifugation for dry
biomass determination. The supernatant was
immediately frozen for further determination of the
lactic acid, glucose, fructose and sucrose concentrations
(Mercier and Yerushalmi , 1992).
to glucose-1-phosphate and fructose. The sucrose was
also by acidic condition, to glucose and fructose
(Zubay, 1984), so the concentration of glucose and
fructose increase. This indicates that the rate of
hydrolysis is faster than fermentation process. The
glucose consumption is better than fructose present in
medium, but both sugars were not completely
utilized. This may reflect the complex nature of
pineapple waste which inhibit the fermentation
process.
The production of lactic acid after 7 days is
54.3 g/l or 79% yield based of the total sugar.
Lactic acid and sugar concentration (g/l)
medium. Anaerobic condition was produced by flushing
with nitrogen and sealing them with thigh-fitting rubber
stopper (Sakamoto and Komagata, 1996). The inoculum
was shaken in incubator shaker at 37 oC, 150 rpm for 24
hours.
(Abdullah)
10
0
0
100
200
0.0
300
Tim e (h)
Figure 1. The Time dependence of biomass, sugar
and lactic acid concentration during fermentation of
liquid waste. Experimental conditions: T: 40oC; pH:
6; 5% inoculum and 50 rpm.
Lactic acid production using solid waste
Solid waste (10%) is extracted by water for
one hour in Erlenmeyer shaker at 250 rpm and room
temperature. The solid particles were separated by
vacuum filter and the liquid was used to fermentation
process in fermentor at 40oC, pH 6, 50 rpm and
inoculum 5%. The initial sugar concentrations about
23.33 g/l of sugar consist only glucose and fructose.
After 6 days the concentration of sugar decreases
from 10.26 to 1.24 g/l and fructose from 12.84 to
4.45 g/l (Fig.2). This indicates that glucose utilization
better than fructose present in medium, but both is
sugars were not completely utilized (87.9 % and
65.34%). The lactic acid production is only 13.1 g/l
or 56% yield. This may reflect that the complex
nature of waste inhibits the fermentation process.
Reaktor, Vol. 11 No.1, Juni 2007, Hal. : 50-52
Rogers P.L. Breach Science Publishers. New York.
pp: 261-277.
16
14
Chandapillai, M.M. and .Selvarajah T., (1978), ”The
Potential Use of Agricultural Waste in Livestock
Production in Malaysia”, Food and Agriculture
Malaysia 2000, Ed. Chin H.F., Faculty of Agriculture
UPM Malaysia. pp: 257-274. 1978.
Concentration (g/l)
12
10
Biomass
Glucose
8
Kroyer, G.T., (1991), “Food Processing Wastes”,
Bioconversion of Waste Material to Industrial
Product. Ed. Martin, A.M. Elviser Applied Science
London. pp:293-303.
Fructose
L. Acid
6
4
Lazaro, M.J., (1989), “Liquid Chromatographic
Determination of Acid and Sugar in Homolactic
Cucumbar Fermentation”, Journal AOAC. 72(1): pp
52-55.
2
0
0
100
200
300
Time (h)
Figure 2. The time dependence of sugar and lactic acid
concentration during fermentation of liquid extract.
Experimental conditions: T: 40oC, pH: 6,5% inoculum
and 50 rpm. .
Conclusions
The chemical composition of the pineapple waste
appears to be a good nutrient for cultivation of lactic
acid bacteria. It can potentially be used as carbon source
for lactic acid fermentation. The yield of lactic acid
from liquid are found to be 79 % yield, while only 56%
yield was produced from solid waste.
References
Abdullah, M. Busairi, and Mat, H.B., (1998), “Pencirian
Bahan Sisa Nanas Tempatan”, Simposium Kimia
Analisis ke Sebelas, Universiti Technologi Malaysia.
Aeschlimann, A. and Stockar, U.V., (1987), “The
Production of Lactic Acid from Whey Permeate by L.
helviticus.” Abstract of Fourth European Congress on
Biotechnology, 2: pp 132-135.
Atkinson ,B., and F. .Mavituna, (1991), ”Biochemical
Engineering and Biotechnology Hand Book“ 2nd.
Edition , Stockton Press, New York, 1181 - 1183.
Buckle, K.A., (1989), “Biotechnology Opportunities in
Waste Treatment and Utilization for The Food
Industry”, Biotechnology and The Food Industry, Ed.
Lund, B., Norddahl, B and Ahring, B., (1992),
“Production of Lactic Acid from Whey Using
Hydrolysed Whey Protein as Nitrogen Source”,
Biotechnology Letters. 14(9): pp : 851-856
Mercier, P. and Yerushalmi, L., (1992), “Kinetics of
Lactic Acid Fermentation on Glucose and Corn by L.
amylophilus”, J. Chem Tech. Biotechnology . 55 : pp
111 - 121.
Milson, P.E., (1987), “Organic Acid by Fermentat
ion”, The Food Biotechnology, Ed. King, R.D.
Elsevier Applied Science. London. pp: 294-298.
Parajo, C.Alonso, J.L and Santos,V., (1996), “Lactic
Acid from Wood”, Process Biochemestry. 31(3).
pp:271-280.
Sakamoto,M. and Komagata, K., (1996), “Aerobic
Growth of An Activities of NADH Oxydase and
NADH Peroxidase in Lactic Acid Bacteria”, J.
Fermentation and Bioengineering, 29 : pp 591 - 602.
Sasaki, K., Noparatnaraphorn, N. and Nagai. S.,
(1991), “Use of Photosynthetic Bacteria for The
Production of SCP and Chemicals from Agro
Industrial Waste”, Bioconversion of Waste Material
to Industrial Product, Ed. Martin. A.M. Elviser
Applied Science. London. pp 225-233.
Zubay, G.L., (1984), “Biochemestry”, USA:
Addison-Wesley Publiushing Company. pp 283-290.
52